Figure 4.

L290P mutation leads to a decrease in S189 phosphorylation of ERK3 protein, whereas L290V mutation has no clear effect. (a) Characterization of a phospho-S189 specific ERK3 antibody. 293 T cells were transfected with wild type ERK3 (ERK3 WT) or ERK3 S189A. Total cell lysates were treated with or without λ phosphatase (PPase). Phosphorylation of ERK3 at S189 [p-ERK3 (S189)] and expression level of ERK3 were determined using a phospho-S189 specific ERK3 antibody and an ERK3 antibody, respectively. (b and c) Western blot analysis of ERK3 phosphorylation at S189 in HeLa cells (b) and in A549 cells (c). Cells were transected with a pSG5 empty vector, HA-tagged wild-type ERK3, or each of the ERK3 mutants as indicated. Two days post-transfection, cells were lysed and levels of total ERK3 and ERK3 phosphorylated at S189 were analyzed by Western blotting. β-actin was used as a loading control.